Damage or infection in the CNS triggers microglia to undergo directed migration towards dead or dying neurons, where they discriminately engulf and eliminate them. This process is tightly controlled in order to sustain the least possible collateral damage to healthy, adjacent neurons. Where there is a loss of this control, microglia are highly reactive to dying neurons and provoke secondary neurotoxicity and as a result are implicated in nearly all CNS neuropathological processes (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, HIV-related dementia, Creutzfeld-Jakob disease). This extreme difference in behavior underscores the importance of understanding the specific signaling systems that both recruit and instruct microglia to selectively adapt their characteristics in response to damaged/dying neurons. We recently demonstrated that N-arachidonoyl glycine (NAGly), an endogenous lipid produced throughout the CNS, drives migration of BV-2 microglial cells more potently than any known endogenous lipid likely through activation of GPR18 receptors. The following proposal will examine the hypotheses that 1) damage to the CNS causes release of NAGly, which drives directed microglial migration in vivo, 2) NAGly signaling phenotypically modifies microglia to perform specialized roles at sites of damage, and 3) the neuronal to microglial communication regulating these actions is initiated through NAGly signaling at GPR18. This will be achieved through three specific aims. Specific Aim 1: Does NAGly drive the activation and migration of CNS microglia? To test the in vivo efficacy and specificity of NAGly-directed migration by immunohistochemistry, we will use pharmacological interventions with a bilateral common carotid artery occlusion (BCCAO) model that drives microglial infiltration in the retina. We will also measure production of NAGly and related lipids in the retina by HPLC/MS/MS analysis to determine the signaling time course of these lipids associated with microglial infiltration. Specific Aim 2: What microglia phenotype does NAGly specify? BV-2 microglia cells stimulated with NAGly drives migration, MAPK activation, and cell proliferation. Here, using primary mouse microglia and mouse cytokine microarray assays (US Biomax) we will determine if NAGly (and related lipids), likewise, initiate pro- or anti- inflammatory phenotypes by quantifying 14 different cytokines. ICC will be used to determine phenotypic changes in morphology. ICC will be used to determine phenotypic changes in morphology. Finally, levels of prostaglandin production will also be measured via HPLC/MS/MS. Specific Aim 3: Does NAGly induce microglial migration and initiation of CNS repair through GPR18? We will use GPR18 knockout mice to 1) measure directed microglial migration in vivo (WT verses KO) and 2) generate a strain of CX3RC1+/GFP/GPR18 KO mouse for future studies of microglia-driven CNS pathophysiologies. These studies of NAGly-GPR18 receptor signaling supply a novel route for understanding neuronal-microglial communication to exploit for potential therapeutics aimed at suppressing dysregulated microglial activity.